IGEM:MIT/2007/Notebook/2007-7-16

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(Status of Expected Parts)
Line 30: Line 30:
*used 4µl of DNA since each was approximately 30ng/µl
*used 4µl of DNA since each was approximately 30ng/µl
*Order Number 8046
*Order Number 8046
 +
 +
==Annealing Complementary Primer Inserts==
 +
*10 uL of 10 uM of oligo
 +
*2 oligos - total volume 20 uL
 +
*Mix with 80 uL water
 +
*PCR Thermocycler Temperatures
 +
*95 C
 +
*Ramp down
 +
*0.1 C/sec

Revision as of 11:06, 16 July 2007

Contents

F2620 Expression Protocol

  1. Create Liquid Cultures in LB Media
  2. Dilute Back 1:500 in LB Media
  3. Grow up for an hour in warm room roller
  4. Split culture in two
  5. Add AHL to one culture to a final concentration of 1E-6M
  6. Grow up for 4 hours in warm room roller
  7. Spin Down, Decant, and Resuspend in PBS
  8. Measure absorbance at 600nm and red fluorescence level

See characterization data for BBa_F2620.

Today's Agenda

  • Redo Overgrown Liquid Cultures
  • Send for Sequencing
  • Cut CPX and Backbone for ligation
  • Start designing high copy backbone
  • PCR off relevant portion of Mer operon from pto40
  • Organize fridge
  • Design and order EC_20 Insert
  • Email Isadora + Tom Knight for Meeting Times--Toan--Done
  • Anneal FHUA Insert oligos

Status of Expected Parts

  1. IDT Primers + Inserts - Arriving Today--ARRIVED
  2. CPX - At FedEx Facility in Boston
  3. FhuA - ARRIVED

Sequencing of LC#1&2 from 3A

  • Miniprepped LC's of those colonies that grew only on Kan plates and not on Kan/Amp plates
  • Sent in 4 samples, one forward and one reverse for each LC
  • used 4µl of DNA since each was approximately 30ng/µl
  • Order Number 8046

Annealing Complementary Primer Inserts

  • 10 uL of 10 uM of oligo
  • 2 oligos - total volume 20 uL
  • Mix with 80 uL water
  • PCR Thermocycler Temperatures
  • 95 C
  • Ramp down
  • 0.1 C/sec
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